The molecular events involved in the translocation of a protein through a biological membrane and the components which mediate this process are just being defined in different membrane systems. Recent advances in the analysis of the mitochondrial import pathway in yeast now provide a well defined system in which detailed biochemical and genetic analysis is possible. The level of molecular analysis defined in the proposed studies should provide new insights into the general problem of intracellular protein transport. Our studies and those of others have demonstrated that the mitochondrial import apparatus consists of components which recognize unique structural features of a mitochondrial import signal as well as an apparatus for reorginization of protein structure to initiate or maintain the import process. The proposed studies in yeast, Saccharomyces cerevisiae will utilize the combination of biochemical fractionation and genetic analysis to define the mitochondrial and cellular components which participate in the reorganization and translocation of mitochondrial precursors from the cytoplasm. Components of the mitochondrial import site will be isolated and characterized by taking advantage of the copurification of translocation intermediates and hybrid mitochondrial precursors which have initiated but not completed protein import. Components of the import site which mediate the unfolding or reorganization of mitochondrial precursors on the mitochondrial surface will be purified using a protein unfolding assay. In all cases isolated components will be used to prepare specific immunological and genetic probes for in vitro and in vivo studies. The yeast mutant MAD1 which exhibits a conditional block in protein import will be further characterized to define its position in the import apparatus and to select pseudorevertants which genetically uncover other components of the import pathway. These pseudorevertants as well as probes obtained by the biochemical approach should facilitate our understanding of the molecular events of protein sorting and transport.